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Open Access Highly Accessed Research

Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum

Pablo Ravasi12, Salvador Peiru1, Hugo Gramajo1 and Hugo G Menzella1*

Author Affiliations

1 Genetic Engineering & Fermentation Technology. Instituto de Biología Celular y Molecular de Rosario-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina

2 Geneg SRL, Cuba 4710, Buenos Aires, Argentina

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Microbial Cell Factories 2012, 11:147  doi:10.1186/1475-2859-11-147

Published: 7 November 2012

Abstract

Background

Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory.

Results

A plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional “fine-tuning”of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering.

Conclusions

We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

Keywords:
Synthetic biology; Metabolic engineering; Corynebacterium glutamicum